The lipid bilayer is the basic structure common to biological membranes. A large fraction of all proteins are associated with membranes, and, as a class, these constitute a huge and diverse target for drug development. Fluidity is critical for biological functions that depend upon the lateral association or clustering of multiple components as well as for processes that change membrane topology such as endo- and exocytocis and fusion. This proposal outlines new types of experiments that probe these two basic aspects of membrane dynamics: the mechanism of vesicle fusion (Aim 1) and the lateral association of certain lipids and membrane anchored proteins into rafts (Aim 2). Both aims depend upon the development of new supported lipid bilayer architectures and analytical methods that can have a broad impact on studies of biological membranes. Aim 1 - Vesicle fusion mechanisms using mobile tethered vesicles: Vesicles can be tethered to fluid supported bilayers and are free to diffuse parallel to the plane of the supported bilayer. The trajectories and collisions of individual tethered vesicles can be visualized by video microscopy. Vesicles displaying different proteins, such as the SNARE proteins, will be tethered at different locations on the supported bilayer, and their subsequent diffusion, docking, hemifusion and fusion can then be monitored in real time at the level of individual vesicles. This new assay system is ideally suited for critical tests of the hypothesis that SNARE proteins are necessary and sufficient for fusion. New methods are proposed for preparing very well defined populations of tethered vesicles displaying specific numbers of v- and t-SNAREs, so that the threshold numbers of cognate proteins needed for each step can be established. Other protein co-factors will then be investigated. Aim 2 - Correlated motion in rafts - composition analysis with 30 nm lateral resolution: The concept of lipid rafts is a useful theme for explaining the origin of specific associations within membranes, despite the overall fluidity of the membrane. Rafts have proven to be quite difficult to study as inter-component correlations may be transient and/or extend over distances that are smaller than the diffraction limit of optical microscopy. We propose to develop new methods to characterize the composition of membranes with very high spatial resolution by multi-isotope imaging mass spectrometry (MIMS). MIMS can be used to determine the composition of a single layer of molecules with a lateral resolution of about 30 nm and extraordinary sensitivity, with the identities of the components encoded by isotopic substitution. Using this method it should be possible to establish the proximity and lateral composition variations of lipids, glycolipids and membrane anchored proteins with unprecedented lateral resolution and without the use of fluorescence labels.